All references, including patents and patent applications, are hereby incorporated by reference in their entireties.
S-Adenosylmethionine (SAM) is a ubiquitous metabolic intermediate that is biosynthesized by the enzyme methionine adenosyltransferase (SAM synthetase), which accelerates the coupling of adenosine triphosphate (ATP) with methionine (Mato, J. M., Pharmacology and Therapeutics, 1997, 73(3), 265-280). SAM plays a key role in various biochemical processes such as enzymatic reactions that involve transmethylation, transsulfuration, and the polyamine-generating aminoalkylation pathway (Roje, S., Phytochemistry, 2006, 67(15), 1686-1698; Giulidori, P. et al., The Journal of Biological Chemistry, 1984, 259(7), 4205-4211). Other enzymatic reactions that involve interaction of proteins with SAM or isostructural SAM analogs include transfer of methylene, ribosyl, and 5′-deoxyadenosyl groups; formation of redox intermediate 5′-deoxyadenosyl radical; and SAM decarboxylation. SAM-nonenzymatic protein interactions also exist wherewith SAM acts as a ligand affecting structural and functional modification in the effector protein (Kozbial, P. Z. and Mushegian, A. R., BMC Structural Biology, 2005, 5, 19-44).

One of the most understood processes involving SAM-utilizing proteins is biochemical transmethylation. The relatively unreactive thioether methyl group of methionine is made very reactive toward nitrogen, oxygen, sulfur, and carbon nucleophiles when coupled with the adenosyl group to provide the chemically destabilizing positively-charged sulfonium ion of SAM. SAM-utilizing methyltransferases are enzymes that catalyze transfer of the reactive methyl group from SAM to a substrate for covalent modification, leaving the stabilized S-adenosylhomocysteine (SAH or AdoHcy) by-product. Methyltransferases comprise a significant percentage of the proteome and are found in all organisms (Petrossian, T. C. and Clarke, S. G., Molecular and Cellular Proteomics, 2011, 10(1)). A large and diverse set of SAM-utilizing methyltransferase substrates is known. Broad substrate classes include histone and non-histone proteins, nucleic acids, polysaccharides, lipids, small organic molecules (e.g. catechol: Mannisto, P. T. and Kaakkola, S., Pharmacological Reviews, 1999, 51(4), 593-628), and inorganic substrates (e.g. arsenic: Thomas, D. J. et al., Experimental Biology and Medicine, 2007, 232(1), 3-13; Hayakawa, T. et al., Archives of Toxicology, 2004, 79(4), 183-191); and halides: (Ohsawa, N. et al., Bioscience, Biotechnology and Biochemistry, 2001, 65, 2397-2404; Attieh, J. M. et al., 1995, 270, 9250-9257). SAM-utilizing methyltransferases play a role in critical cellular processes including biosynthesis, signal transduction, chromatin regulation, and gene silencing.
While the SAM-utilizing methyltransferases share a common requirement for SAM, distinct differences exist in the SAM binding structural fold and also in the SAM binding mode. These different structural families can be grouped into at least seven classes, five classes that are typically designated I through V (Schubert, H. L. et al., Trends in Biochemical Sciences, 2003, 28(6), 329-335). Two other classes include the radical SAM enzymes (Frey, P. A. et al., Critical Reviews in Biochemistry and Molecular Biology, 2008, 43, 63-88), which catalyze diverse radical-based reactions that include methylation, and the isoprenylcysteine carboxy methyltransferases (ICMTs), which are integral membrane proteins (Yang, J. et al., Molecular Cell, 2011, 44(6), 997-1004). Amino-acid sequence homology within each class can be as low as 10%, showing that wide variations in molecular environment mediating catalysis of methyl transfer from SAM to substrate are allowed, which at least in part may be due to the favorable energetics involved in the conversion of SAM to SAH. Methyltransferases that bind and methylate protein substrates are generally found in Class I (classical fold) or Class V (SET fold), and methyltransferases that act on DNA substrates have been found in Classes I or IV (Schubert, H. L. et al., Trends in Biochemical Sciences, 2003, 28(6), 329-335). Non-catalytic domains outside the core structure determine substrate recognition. SAM-utilizing methyltransferases recruit SAM and a substrate to the SAM-dependent methyltransferase fold, where methyl transfer occurs and modified substrate and SAH are produced and released.
The involvement of methyltransferases in epigenetics is currently an intense area of research (Copeland, R. A. et al., Nature Reviews Drug Discovery, 2009, 8, 724-732). The field of epigenetics studies molecular changes such as DNA methylation and post-translational histone modifications that influence phenotype without alterations in the DNA sequence of the genome. Methyltransferases play a major role in epigenetic regulation of gene expression by catalyzing the modification of chromatin by specific methylations of DNA, histones, or biomolecules associated with chromatin (Kouzarides, T., Cell, 2007, 128, 693-705). Chromatin remodeling methyltransferases can generally be divided into two categories according to their substrates. DNA methyltransferases (DNMTs) methylate the 5-position carbon atom of cytosine in the CpG dinucleotide sites of the mammalian genome (Cheng, X. and Blumenthal, R. M., Structure, 2008, 16, 341-350). Protein methyltransferases (PMTs), which can generally be subdivided into protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs), methylate protein lysine or arginine residues, respectively. The histone targets of PMTs are largely characterized while non-histone protein targets such as the FOXO transcription factors continue to be discovered (Yamagata, K. et al., Molecular Cell, 2008, 32, 221-231; Greer, E. L. and Brunet, A., Oncogene, 2005, 24, 7410-7425).
Aberrant DNA and histone methylation due to abnormal methyltransferase expression levels or mutations is associated with the onset and progression of a variety of cancers and other diseases and conditions (Egger, G., Nature, 2004, 429, 457-463; Esteller, M., New England Journal of Medicine, 2008, 358, 1148-1159).
Selective, small-molecule inhibitors of epigenetic targets, such as histone deacetylases (HDACs) and DNMTs, have been successfully designed and deployed as therapeutic agents for the treatment of myelomas, lymphomas, and other cancers and have been further investigated for use against inflammatory and autoimmune disorders (Copeland, R. A. et al., Nature Reviews Drug Discovery, 2009, 8, 724-732; Szyf, M., Clinical Reviews in Allergy and Immunology, 2010, 39(1), 62-77; Kaiser, J., Science, 2010, 330(6004), 576-578). PKMTs and PRMTs are favorable drug targets amenable to small-molecule inhibition (Copeland, R. A. et al., Current Opinion in Chemical Biology, 2010, 4, 505-510). Each methyltransferase is structurally unique and has a distinct functional profile (Dillon, S. C. et al., Genome Biology, 2005, 6, 227; Cheng, X. et al., Annual Reviews in Biophysical and Biomolecular Structure, 2005, 34, 267-294). Small molecule inhibitors are currently sought after for a variety of methyltransferases in the search for new drug therapies (Shaaban, S. A. and Bedford, M. A., Chemistry and Biology, 2007, 14(3), 242-244), and the potential pharmaceutical utility in areas such as antibiotics and treatment of Parkinson's Disease (e.g. catechol-O-methyltransferase inhibitors) and beyond is vast; thus, an efficient means for rapidly screening large compound collections against an ever-growing number of known SAM-utilizing methyltransferases is needed.
Various SAM-utilizing methyltransferase screening assays have been developed and used for identifying compound inhibitors. One such assay reported is a universal competitive fluorescence polarization (FP) methyltransferase activity immunoassay that measures formation of SAH (Graves, T. L. et al., Analytical Biochemistry, 2008, 373, 296-306). The assay employs an anti-AdoHcy antibody and fluorescence-labeled AdoHcy conjugate tracer to measure AdoHcy generated from the methyltransferase activity. Another SAM-utilizing methyltransferase assay reported is an enzyme-coupled continuous spectrophotometric screen (Dorgan, K. M. et al., Analytical Biochemistry, 2006, 350, 249-255). In this assay SAH generated from demethylation of SAM is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Adenine is subsequently hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase, a process which is monitored continuously by measuring absorbance at a wavelength of 256 nm. Another enzyme coupled assay used for measuring SAM-utilizing methyltransferase activity involves the conjugation of homocysteine (Hcy), which is generated from cleavage of SAH by SAH hydrolase (SAHH), to a thiol-reactive fluorophore (Collazo, E. et al., Analytical Biochemistry, 2005, 342, 86-92). A commercial radiometric histone methyltransferase assay is also known and has been adapted for high throughput screening (Horiuchi, K. Y. et al., FASEB J, 2010, 24, lb61). All of these assays gauge SAM-utilizing methyltransferase activity by measuring the generation of products formed as a consequence of the signature methyl-transfer reaction from SAM to the substrate but do not directly provide information as to the specific binding interactions of the test compound without subsequent enzymological study. In addition, target screening using a coupled enzyme assay method suffers from the potential generation of false positive leads due to inhibition of the coupling enzymes. The radiometric assay is a robust binding assay but possesses the inherent liability of generating radioactive waste. The assays developed here overcome these shortfalls and will also provide specific binding information by directly measuring binding affinities and dissociation constants of the test compounds.
A fluorescence polarization or TR-FRET assay could measure test compound binding to a SAM-utilizing protein by measuring displacement of a fluorescence-labeled ligand (“detection analyte” or “probe”) from the protein. The universal cofactor SAM provides a structural template with which to design a versatile detection analyte. SAM itself is a chemically reactive methyl donor and thus is not a suitable compound for incorporation into a stable detection analyte. A robust, chemically stable SAM mimic possessing steric and electronic characteristics similar to those of SAM is therefore desirable. Such an analyte design would provide a SAM-utilizing protein ligand moiety seeking to take advantage of inherent pan-methyltransferase recruitment of SAM and could thus allow the analyte to be utilized across the SAM-dependent methyltransferase enzyme family. Herein are disclosed fluorescent detection analytes, assays that employ them, and their uses for assessing binding of test compounds.
Sinefungin, a SAM- and SAH-analog nucleoside isolated from Streptomyces griseolus and Streptomyces incarnatus, exhibits an array of antimicrobial effects due primarily to its inhibition of SAM-utilizing methyltransferases (Malina, H. et al., Journal of Antibiotics, 1985, 38(9), 1204-1210; Berry, D. R. and Abbott, B. J., Journal of Antibiotics, 1978, 31(3), 185-191; Vedel M. et al., Biochemical and Biophysical Research Communications, 1978, 85(1), 371-376). The sinefungin molecular structure may be divided into three subunits: a central ribose ring, an adenine ring connected by its 9-nitrogen position to the 1′-ribose ring carbon atom, and an ornithine side chain connected by its amino acid δ-carbon to the 5′-carbon atom of the ribose ring. Recent reports disclose structures of sinefungin bound in the SET domain of histone PKMTs SET7/9, LSMT, SmyD1 and SmyD3 (Subramanian, K. et al., Molecular Cell, 2008, 30(3), 336-47; Couture et al., The Journal of Biological Chemistry, 2006, 281(28), 19280-19287; Sirinupong, N. et al., The Journal of Biological Chemistry, 2010, 285(52), 40635-40644; Sirinupong, N. et al., Journal of Molecular Biology, 2010). Earlier published sinefungin-nucleic acid methyltransferase complex structures wherein sinefungin is shown to bind in the SAM binding site show only minor structural changes in the complex versus the cases in which SAM or SAH are shown bound (Zheng, S. et al., The Journal of Biological Chemistry, 2006, 281(47), 35904-35913; Thomas, C. B. et al., The Journal of Biological Chemistry, 2003, 278(28), 26094-26101).

SAM-like affinity for a wide range of methyltransferases and relative chemical stability make sinefungin a reasonable detection analyte SAM-utilizing protein methyltransferase ligand moiety. For example, sinefungin binds to human SET7/9 with only a six-fold lower affinity than SAM, and sinefungin binds to Arabidopsis LSMT with only a fourteen-fold lower affinity than SAM (Couture et al., The Journal of Biological Chemistry, 2006, 281(28), 19280-19287; Horowitz et al., The Journal of Biological Chemistry, 2011, Epub jbc.M111.232876). Variations in spatial requirements among different methyltransferase catalytic domains may require that an assortment of sinefungin-based detection analytes be utilized to screen test compounds against a broad panel of these enzymes. Sinefungin-based detection analyte alternatives may vary based on different linker-fluorophore attachment positions on sinefungin. The linker moiety, for example, may be covalently bound to sinefungin at a nitrogen or carboxy oxygen position on the ornithine residue, one of the two ribose hydroxyloxygen atoms, or to an open carbon or the free amino position on the C-6 carbon atom of the adenine base ring. Sinefungin-based probes may be designed that are selective to SET domain-containing lysine methyltransferases or other classes of methyltransferases, which can use different binding modes to recognize SAM. These differences may necessitate that the linker moieties tethering sinefungin or sinefungin analogs to the fluorophore be attached to sinefungin at different positions to accommodate the varying binding modes.
Fluorescence polarization and TR-FRET assays generally provide advantages in the study of protein-ligand binding over other conventional assay types. These assay formats allow rapid real-time measurements, avoid the use of radioactive materials, are homogeneous requiring minimal additions and no washing steps, and may possess sub-nanomolar detection limits. FP and TR-FRET assays are currently used in drug discovery and are routinely converted to high-throughput screening (HTS) formats (Burke, T. J. et al., Combinatorial Chemistry and High Throughput Screening, 2003, 6(3), 183-194). The uses, advantages, and photophysical principles associated with FP and TR-FRET assays have been described and are well known to those ordinarily skilled in the art (Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Springer, N.Y., USA, 1999; Owicki, J. C., Journal of Biomolecular Screening, 2000, 5(5), 297-306; Nasir, M. S., Jolley, M. E., Combinatorial Chemistry & High Throughput Screening, 1999, 2, 177-190; Klostermeier, D. and Millar, D. P., Biopolymers (Nucleic Acid Sciences), 2002, 61, 159-179).